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An Efficient Androgen Response, Antioxidant Defense and Proteosomal Pathway Maintains Fertility in Donors with ROS Positive

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An Efficient Androgen Response, Antioxidant Defense and Proteosomal Pathway Maintains Fertility in Donors with ROS Positive AN EFFICIENT ANDROGEN RESPONSE, ANTIOXIDANT DEFENSE AND PROTEOSOMAL or Reprod er f uc nt ti e ve C M n a e PATHWAY MAINTAINS FERTILITY IN DONORS WITH ROS POSITIVE SPERM d c i i r c e i n m e A Luna Samanta, PhD1, Ashok Agarwal, PhD2, Rakesh Sharma, PhD2, Narasimhan Kothandaraman, PhD2 and Nirlipta Swain, MS1 C e 3 n 9 t 9 ru . 1 m st 1 Ex e E Redox Biology Laboratory, School of Life Science, Ravenshaw University, Orissa, India, cellentia 2American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH ABSTRACT MATERIALS and METHODS RESULTS Objective: Reactive oxygen species (ROS) in spermatozoa involves electron leakage All specimens were collected by masturbation at the Andrology Laboratory of Cleveland Clinic after 2-3 days of A. B. from the sperm mitochondria resulting in organelle damage and initiation of intrinsic sexual abstinence. Institutional Review Board of Cleveland Clinic approved the entire protocol used in the study. Negative Threonine Cellular homeostasis2% apoptotic cascade. As a consequence spermatozoa lose their motility, vitality, DNA protease regulation of Signal 3% Semen samples were collected from fertile donors (n=17). Semen analysis was performed. Leukocytospermia was 3% ligase activity Defense response integrity and subsequently undergo apoptosis. Apoptosis results in the externalization of 6% 3% 6 Catalytic Positive regulation Melanosome recorded when the round cell concentration was >1 X 10 /mL in the sample. It was confirmed by the peroxidase activity of catalytic activity 6% phosphatidylserine, which may facilitate the silent phagocytosis of these cells following 7% 5% 6 Glycoprotein Vesicle 7% insemination, in turn influencing the female tract immune response to sperm antigens and or the Endtz test. Specimens that were positive for the Endtz test (>1×10 white blood cells/ mL) were not included in the study. 8% Annexin Phosphorylation Proteolysis 2% subsequent fertility. Therefore, in this study, an attempt has been made to answer how 1% 5% Immune effector process Metal ion Signal peptide Acetylated amino Reactive Oxygen Species (ROS) Measurement 1% some men with high ROS level remain fertile utilizing high throughput proteomic analysis. binding 7% end 6% Cell cycle ROS formation was measured by chemiluminescence assay in the semen using 5 mM luminol (5-amino-2,3- 4% Protein 3% complex Design: Proteomic analysis of spermatozoa from ROS positive and negative fertile donors. Defense response biogenesis dihydro-1, 4-phthalazinedione) as the probe continuously for 15 min using a Berthold luminometer (AutolumatPlus 953, 2% 2% Calcium ion binding 5% 6 Cell adhesion Oxidoreductase Lipid Oakridge, TN). ROS positive samples were furthergrouped into low ROS <500 RLU/sec/10 sperm and High ROS >500 2% 5% Protein localization binding Materials and Methods: Spermatozoa from fertile men with low ROS levels (control 4% 2% 6 Secretion 6 Protein complex 2% RLU/sec/10 sperm. Chaperone group, n=17, <500RLU/s/10 sperm) and high ROS levels (experimental group, biogenesis Glycolysis 2% Glycolysis 4% 6 2% Pyruvate metabolism 1% 1% n=14, (>500 RLU/s/10 sperm) were used in this study. Following normalization Identical protein Response Sample Preparation and Global Proteomics Analysis binding Cell motion of protein concentration, samples were pooled, and proteins were extracted and 3% to Cell projection oxidative 3% 3% Stress response Regulation of apoptosis stress Samples were pooled from 4 subjects in each group for proteomic analysis. Only those samples that had equal amount 3% separated by 1-D gel (n=3). Bands were digested and loaded on a LTQ-Orbitrap 2% 1% Proteolysis 3% Nucleotide binding Elite hybrid mass spectrometer system. Functional annotations of proteins were Response to Sexual reproduction 4% 9% of protein contributed by similar number of spermatozoa in each patient and the fertile group were used. Equal oxidative stress 2% Cell cycle obtained using bioinformatics tools and pathway databases. Ion Regulation of ligase amounts of proteins from each group were resolved on a 1D SDS-PAGE. After in gel digestion extracted peptides Spermatogenesis 2% activity 3% homeostasis Regulation of 2% 1% Cell Cofactor binding 3% Oxidative cell death were resuspended in a final volume of ~30 µL for LC-MS analysis. All MS/MS samples were analyzed using 5% motility phosphorylation 2% Disulfide Carbohydrate Results: Our results show overexpression of ROS generating system as a function of bioysnthetic process 2% Mascot, SEQUEST and X! Tandem. bond Generation of 7% Secreted 1% Sexual Homeostatic Anti-apoptosis Glycoprotein Microtubule precursor metabolites enhanced mitochondrial activity as evidenced by overexpression of transcript variants 5% reproduction process 3% 7% based process 3% and energy 3% of NADH dehydrogenase, NDUFS 3 and 5 were overexpressed in the high ROS group. Criteria for Protein Identification 2% 4% The oxidative predominance is balanced by enhanced antioxidant defense where To validate MS/MS-based peptide and protein identifications was done by Scaffold (established at >95.0% C. regulation of D. both cytosolic and mitochondrial superoxide dismutase (SOD1 and 2), glutathione transcription (6) Sexual probability and 99% for protein) by the Peptide Prophet algorithm with Scaffold delta-mass correction to achieve metal ion reproduction peroxidase Mu 3 and omega 2, mitochondrial glutathione reductase, thioredoxin regulation of binding phosphorylation (22) Mitochondrion reductase 2, peroxidextrin 4, Apolipoprotein A1 and testis specific glyceraldehyde- a false discovery rate (FDR) of <1.0%. Proteins were annotated with gene ontology (GO) terms from National (4) translational elongation (3) Center for Biotechnology Information (NCBI). glucose metabolic process (3) 3-phosphate dehydrogenase (GAPDH) were overexpressed. Moreover, mitochondrial Generation of precursor protein transport (10) metabolites and energy Ion binding Cell motility thioredoxin dependent peroxide reductase and thioredoxin related transmembrane 12% 12% Quantitative Proteomics cell fraction protein 4 were uniquely expressed in high ROS group providing additional defense. (15) oxidative phosphorylation (12) Overexpression of Apolipoprotein D in this group is suggestive of better androgen For proteomic analysis, the relative quantity of the protein was determined by comparing the number of spectra (termed as muscle tissue development (4) Cell adhesion 9% response despite oxidative stress in this group. Pathway analysis of differentially spectral counts SpCs), used to identify each protein. Different constraints for significance tests (p-value) and/or induction of apoptosis (7) expressed proteins indicate that in low ROS group the net protein turnover was electron carrier activity (10) oxidation reduction (23) fold change cutoffs or normalized spectral abundance factor (NSAF ratio) were applied as shown below: Signal Oxidoreductase 23% regulated by an efficient molecular chaperone system with upregulated such as 9% T-complex proteins, HSP 90 and 70 family of proteins along with other assisting i. Very Low abundance: spectral count range 1.7-7; p ≤ 0.001 and NSAF ratio ≥ 2.5 hydrolase (19) coenzyme binding (13) proteins such as ANAX2, P4HB etc. On the other hand, in high ROS group the for overexpressed, ≤ 0.4 for underexpressed proteins cellular homeostasis (8) Regulation of peptidase activity (10) phosphorylation general protein turnover is regulated by an efficient ubiquitin-proteosome pathway 9% ii. Low abundance: spectral count range 8-19; p≤ 0.01 and NSAF ratio ≥2.5 ATPase activity (11) macromolecular complex as evidenced by overexpression of PSMA1, PSMA2, PSMA3, PSMA4, PSMA5, assembly (11) for overexpressed, ≤ 0.4 for underexpressed proteins ion transmembrane transport (5) Disulfide bond PSMA6, PSMA7, PSMB1 and PSMB2 along with proteins involved in maintenance protein complex biogenesis (9) regulation of ligase activity (6), 26% iii. Medium abundance: spectral count range between 20-79; p≤ 0.05 and ER (19), translocation (5) calcium binding EF-hand (7), of morphology and motility of spermatozoa. regulation of apoptosis (15) proteolysis NSAF ratio ≥ 2.0 for overexpressed, ≤ 0.5 for underexpressed proteins (15) translation calcium ion (11) fatty regulation binding iv. High abundance: spectral counts >80; p≤ 0.05 and NSAF ratio ≥ 1.5 acid (11) Conclusions: The results of the present study demonstrate that an efficient androgen metabolism of protein (6) cell cycle response, antoxidant defence and proteosomal pathway is responsible for process ubiquitination for overexpressed, ≤ 0.67 for underexpressed proteins (8) (6) maintaining fertility in ROS positive donor sperms. Bioinformatics Analysis Figure 1. Comparison of enriched expression profile of differentially expressed proteins. A. Overexpressed proteins; Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. B. Underexpressed proteins; C. Proteins unique to ROS + group and D. Proteins unique to ROS - group. INTRODUCTION RESULTS The ability of spermatozoa to generate reactive oxygen species (ROS) is a Expression Profile of Differentially Expressed Proteins universal property of mature spermatozoa from all mammalian species. It 1. Of the 340 differentially expressed proteins (DEPs), 92 were overexpressed, 95 were underexpressed and 16 were uniquely is also a major contributor to the oxidative stress responsible for defective expressed in ROS+ group. sperm function. The mechanisms by which oxidative stress limits the 2. 137 unique proteins were
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